Project Summary/Abstract Development is remarkably reproducible, generally producing the same organ with invariant size, shape, structure, and function in each individual. Robustness is the ability of cellular systems to adjust and develop the correct size and shape organs in the face of stochastic fluctuations, and environmental and genetic variations. In the absence of robustness, these perturbations would cause large variations in organ size and shape. The mechanisms proposed to create robustness include negative feedback loops, redundancy in regulatory networks, intercellular signaling to coordinate cell behaviors, spatiotemporal averaging of variability, and mechanical signaling, all of which function at the systems level and are repeatedly observed in multicellular organisms including humans. To determine how robustness emerges, we identified mutants with enhanced variability in organ size or shape (vos), thus disrupting robustness. Our Arabidopsis thaliana sepal (outermost floral organ) system is uniquely well suited for these studies because each plant produces more than 100 sepals, allowing statistical analysis of organ robustness in a single individual, which cannot be done in most other systems. Our goal is to use these vos mutants in combination with an innovative quantitative biology approach combining live imaging of growing sepals, image processing to quantify growth, computational modeling, biomechanical tests, molecular genetics, and genomics to elucidate mechanisms generating robustness. We hypothesize that mechanical signaling and coordination of growth may be particularly important for robustness of organ shape and size. (Aim 1) First, we will elucidate the role of mechanical signaling in spatiotemporal averaging to produce robust organs. Based on computational modeling, we hypothesize appropriate mechanical signaling is required for spatiotemporal averaging of cellular growth variability to produce robust organs. We will test this hypothesis using mutants that alter mechanical signaling in conjunction with the vos1 mutant, which inhibits spatiotemporal averaging. (Aim 2) Second, we will determine the mechanisms that synchronize organ initiation and how they contribute to robustness of organ size. vos2 mutants exhibit variable sepal size and irregular timing of sepal primordia initiation. We will test the hypothesis that variable organ sizes result from a loss of robustness in the timing of organ initiation through examining biomechanics and hormone signaling, both of which contribute to primordium initiation. (Aim 3) Third, we will determine how coordination of growth across the three dimensions of the organ contributes to robustness in shape. vos3 mutants exhibit variable sepal shapes; we hypothesize this results from perturbed coordination of growth between tissue layers leading to mechanical buckling of the outer sepal surface. We will use computational modeling to predict differences in growth rate and mechanics that can cause mechanical buckling and test these in vos3. Together, these aims will reveal mechanisms and principles generating robustness of organ size and shape, which underlie human development.